Receptor-bound alpha 2-macroglobulin (alpha 2M) undergoes a two-step process in its internalization by cultured fibroblasts. First, the receptor- alpha 2M complexes concentrate in coated pits on the cell surface. Second, the alpha 2M is internalized into endocytic vesicles we have termed receptosomes. Using a variety of monovalent ionophores and inhibitors of ATP synthesis, the present report provides data that discriminates between these two steps. Appearance of alpha 2M-receptor complexes in coated pits occurs at 4 degrees C and is inhibited by primary amines as well as some other drugs and chemical reagents [1, 2]. Internalization of alpha 2M-receptor complexes into receptosomes is inhibited by monovalent ionophores that disrupt proton gradients (monensin, nigericin, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone, and 3,3',4',5-tetrachlorosalicyanilide), but not the Na+ specific ionophore antamanide or the K+ specific ionophore valinomycin. Using electron microscopy, the proton ionophores appear to interfere with the transfer of alpha 2M from coated pits to receptosomes. Prolonged incubation with monensin in the presence of alpha 2M also decreases the number of alpha 2M receptors on the cell surface, but this did not appear sufficient to account for the extensive inhibition of internalization. Monensin also inhibited the internalization of vesicular stomatitis virus and epidermal growth factor (EGF). Our data suggest that a proton gradient may be necessary for receptor-mediated endocytosis of alpha 2M and some other ligands.